JP2011208993A - Analyzing substrate, and method for manufacturing the same - Google Patents
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Abstract
Description
本発明は、臨床検査、環境モニタリング、食品検査、創薬等に用いることができる分析用基板及びその製造方法に関する。 The present invention relates to an analytical substrate that can be used for clinical testing, environmental monitoring, food testing, drug discovery, and the like, and a method for manufacturing the same.
近年、表面増強効果を利用した光による分析方法が注目を集めている。金属や誘電体、あるいは半導体の微粒子の物理化学的特性は、微粒子の厚さ、サイズ、形状等に大きく依存する。例えば、サイズ１００ｎｍ以下の金、銀、プラチナ等のナノ粒子に光を照射すると、ナノ粒子近傍に近接場と呼ばれる局在した強力な光の場が発生する。測定対象である分子ないしは薄膜状のサンプルを貴金属ナノ粒子でコーティングされた基板に展開して光学的手法にて分析することより、信号が数十倍（蛍光法および赤外分光法）から１００万倍（ラマン分光法）増大されることが知られている。 In recent years, analysis methods using light utilizing the surface enhancement effect have attracted attention. The physicochemical characteristics of metal, dielectric, or semiconductor particles depend greatly on the thickness, size, shape, etc. of the particles. For example, when a nanoparticle such as gold, silver, or platinum having a size of 100 nm or less is irradiated with light, a localized strong light field called a near field is generated in the vicinity of the nanoparticle. A molecule or thin film sample to be measured is developed on a substrate coated with noble metal nanoparticles and analyzed by an optical method, so that the signal is several tens of times (fluorescence and infrared spectroscopy) to 1 million. It is known to be doubled (Raman spectroscopy).
光の表面増強効果を利用したデバイスとして、例えば、表面増強効果によって生じる表面プラズモン共鳴（ＳＰＲ：Ｓｕｒｆａｃｅ Ｐｌａｓｍｏｎ Ｒｅｓｏｎａｎｃｅ）と呼ばれる現象を利用したバイオセンサ等がある。 As a device using the surface enhancement effect of light, for example, there is a biosensor using a phenomenon called surface plasmon resonance (SPR) generated by the surface enhancement effect.
これらの方法の要は、基板上に形成した固相化金属微粒子の作製にあり、適切なサイズ、厚さ、形状等の貴金属ナノ粒子を高密度かつ均一に形成することである。従来の作製方法としては、非特許文献１に示されるように、金属コロイドを基板上に固定する方法、真空蒸着された金属を加熱によりアイランド化する方法、リソグラフィーにより金属をパターニングする方法がある。また、特許文献１に示されるように、基板に吸着させたポリスチレン球上に、真空蒸着により、金属層を形成する方法が提案されている。 The main point of these methods is to produce solid-phased metal fine particles formed on a substrate, and to form noble metal nanoparticles having an appropriate size, thickness, shape and the like with high density and uniformity. As a conventional manufacturing method, as shown in Non-Patent Document 1, there are a method of fixing a metal colloid on a substrate, a method of forming an island by heating a vacuum-deposited metal, and a method of patterning a metal by lithography. Moreover, as shown in Patent Document 1, a method of forming a metal layer on a polystyrene sphere adsorbed on a substrate by vacuum deposition has been proposed.
ところで、上記のように表面増強効果を利用した光学的手法で分析する場合には、使用する光の波長が分析方法によって異なる。したがって、固相化金属微粒子の組成、厚さ、サイズ、形状等を調整し、用いられる光の波長に合わせる必要がある。例えば、あるサンプルをラマン分光法と蛍光分光法の２種類の分光法により分析する場合には、使用する光の波長が異なるため、異なる金属微粒子が形成された分析用基板を２枚用意する必要がある。 By the way, when analyzing by the optical method using the surface enhancement effect as described above, the wavelength of the light used varies depending on the analysis method. Therefore, it is necessary to adjust the composition, thickness, size, shape, etc. of the solid-phased metal fine particles to match the wavelength of the light used. For example, when analyzing a sample by two types of spectroscopy, Raman spectroscopy and fluorescence spectroscopy, the wavelength of light used is different, so it is necessary to prepare two analytical substrates on which different metal fine particles are formed. There is.
この場合、液体もしくは粉末状の分析対象物（サンプル）が大量にある場合には問題ではないが、サンプル量が非常に微量な場合には、両方の分析方法による測定結果が十分なものではなくなる可能性がある。また、組織切片等のように一つのサンプルを２次元マッピングする場合には、どちらかの分析方法を選択しなければならなかった。 In this case, it is not a problem when there are a large amount of liquid or powder-like analytes (samples), but when the amount of samples is very small, the measurement results by both analysis methods are not sufficient. there is a possibility. In addition, when one sample is two-dimensionally mapped, such as a tissue section, one of the analysis methods has to be selected.
また、分析用基板を作製する方法の一つに、島状薄膜蒸着法があるが、この方法では金属粒子を均一に高密度化して形成することは困難である。他方、電子線リソグラフィー（ＥＢＬ：Ｅｌｅｃｔｒｏｎ Ｂｅａｍ Ｌｉｔｈｏｇｒａｐｈｙ）、原子間力顕微鏡リソグラフィー（ＡＦＭＬ：Ａｔｏｍｉｃ Ｆｏｒｃｅ Ｍｉｃｒｏｓｃｏｐｅ Ｌｉｔｈｏｇｒａｐｈｙ）などを用いて基板に金属微粒子を形成する方法などもあるが、非特許文献１に示すように、膨大なコストと低いスループットという大きな問題がある。したがって、分析方法の種類毎に、何枚も分析用基板を作製することは、手間やコストの点からも問題であった。 In addition, as one of the methods for producing the analysis substrate, there is an island-shaped thin film deposition method, but it is difficult to form the metal particles with a uniform high density by this method. On the other hand, there is a method of forming metal fine particles on a substrate using electron beam lithography (EBL), atomic force microscope lithography (AFML), etc., as shown in Non-Patent Document 1. In addition, there is a big problem of huge cost and low throughput. Therefore, it has been a problem from the viewpoint of labor and cost to produce several analysis substrates for each type of analysis method.
また、特許文献１のような手法を用いたとしても、分析方法の種類毎に、何枚も分析用基板を作製することは手間もコストもかかる。 Further, even if a technique such as Patent Document 1 is used, it takes time and cost to produce a number of analysis substrates for each type of analysis method.
本発明は、上述した課題を解決するために創案されたものであり、手間やコストをかけずに、微量のサンプル、もしくは二次元情報を含む組織切片等の同一サンプルを、複数の分析方法で高感度に分析することができる分析用基板及びその製造方法を提供することを目的とする。 The present invention was devised to solve the above-described problems, and a plurality of analysis methods can be used to analyze the same sample such as a small amount of sample or a tissue section containing two-dimensional information without labor and cost. It is an object of the present invention to provide an analytical substrate that can be analyzed with high sensitivity and a method for manufacturing the same.
上記目的を達成するために、本発明の分析用基板は、金属が付着した固相化微粒子が基板上に形成された表面増強効果を有する分析用基板であって、前記固相化微粒子が形成された領域は、光学的分析方法の種類に対応して前記金属の種類もしくは金属の付着量を変えた領域、又は前記金属の種類及び金属の付着量を変えた領域に分割されていることを主要な特徴とする。 In order to achieve the above object, the analytical substrate of the present invention is an analytical substrate having a surface enhancement effect in which solid-phased fine particles to which metal is attached are formed on the substrate, wherein the solid-phased fine particles are formed. The region is divided into a region in which the type of metal or the amount of metal adhesion is changed corresponding to the type of optical analysis method, or a region in which the type of metal and the amount of metal adhesion are changed. Main features.
また、本発明の分析用基板の製造方法は、基板上に微粒子を固相化する第１の工程と、 表面増強効果を利用した光学的分析方法の種類に対応して、前記微粒子の上に付着させる金属量を前記微粒子の固相化領域毎に変える第２の工程とを備えたことを主要な特徴とする。 In addition, the method for producing an analytical substrate of the present invention comprises a first step of solidifying microparticles on the substrate, and an optical analysis method using a surface enhancement effect. And a second step of changing the amount of metal to be deposited for each solid-phase region of the fine particles.
本発明によれば、微量又は唯一無二のサンプルを複数の異なる分析方法により分析することが可能となる。例えば、がん治療における腫瘍部位の診断において、摘出切片中の標的バイオマーカーと化学分析を同時に行うことができれば、より的確な判定が可能になる。 According to the present invention, a trace amount or a unique sample can be analyzed by a plurality of different analysis methods. For example, in diagnosis of a tumor site in cancer treatment, if a target biomarker and chemical analysis in an excised section can be performed simultaneously, more accurate determination can be made.
また、腫瘍切片のように、二次元マッピングが必要な場合においては、同一領域を異なる分析方法で解析することによる情報の高精度化を行えることが有効となる。測定対象が微量でも貴重でもない場合においても、分析手法毎に試料を準備する必要がなくなり、測定の手間を省くことができる。さらに品質管理のような応用においては、分析のスループット向上につながり、全量検査も可能となる。 In addition, when two-dimensional mapping is necessary, such as a tumor slice, it is effective to improve the accuracy of information by analyzing the same region using different analysis methods. Even when the measurement target is neither a minute amount nor precious, it is not necessary to prepare a sample for each analysis method, and the measurement labor can be saved. Furthermore, in applications such as quality control, the analysis throughput can be improved, and the entire quantity can be inspected.
以下、図面を参照して本発明の一実施形態を説明する。構造に関する図面は模式的なものであり、現実のものとは異なる。また、図面相互間においても互いの寸法の関係や比率が異なる部分が含まれている。 Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The drawings relating to the structure are schematic and different from the actual ones. Moreover, the part from which the relationship and ratio of a mutual dimension differ also in between drawings is contained.
図１（ａ）は、本発明の分析用基板の基本的な構成を示す。ガラスやシリコン等、光学的に平坦な基板１の上に、微小球形状の金属付着微粒子１０が、一様に形成されている。金属付着微粒子１０は、例えば、粒径２０ｎｍから１０００ｎｍの微小球形状の微粒子２と微粒子２に付着した金属層３とで構成されている。基板１上に微粒子２が固相化されており、微粒子２の表面の上半分程度が金属層３で被覆されている。金属層３は、金、銀、プラチナ等の金属で構成されている。基板１上の領域によって、金属層３の組成、厚さ、微粒子２の粒径が異なるように構成される。 FIG. 1A shows a basic configuration of the analysis substrate of the present invention. On the optically flat substrate 1 such as glass or silicon, fine spherical metal-attached fine particles 10 are uniformly formed. The metal-attached fine particles 10 are composed of, for example, microspherical fine particles 2 having a particle diameter of 20 nm to 1000 nm and a metal layer 3 attached to the fine particles 2. The fine particles 2 are solid-phased on the substrate 1, and the upper half of the surface of the fine particles 2 is covered with the metal layer 3. The metal layer 3 is made of a metal such as gold, silver, or platinum. Depending on the region on the substrate 1, the composition and thickness of the metal layer 3 and the particle size of the fine particles 2 are different.
分析用基板の領域１１を拡大したのが、例えば図１（ｂ）であり、粒径が同じ微粒子２が用いられているものの、微粒子２上に形成された金属層３の厚さが異なっている。範囲Ａに存在する微粒子と範囲Ｂに存在する微粒子２の粒径は同じであるが、金属層３の厚さは範囲Ａよりも範囲Ｂの方が厚く形成されている。これにより、範囲Ｂの方が微粒子２に付着する金属の量は多くなる。 For example, FIG. 1B is an enlarged view of the region 11 of the analysis substrate. Although the fine particles 2 having the same particle diameter are used, the thickness of the metal layer 3 formed on the fine particles 2 is different. Yes. Although the particle diameters of the fine particles existing in the range A and the fine particles 2 existing in the range B are the same, the thickness of the metal layer 3 is larger in the range B than in the range A. Thereby, the amount of the metal adhering to the fine particles 2 is larger in the range B.
一方、領域１２を拡大したのが、例えば図１（ｃ）であり、粒径が異なる微粒子２が用いられている例を示す。基板１上には、範囲Ｃよりも範囲Ｄの方が粒径が大きい微粒子２が固相化されている。このため、微粒子２上に形成される金属層３の面積は範囲Ｄの方が大きくなる。なお、粒径が異なる微粒子２を用いるということは、言い換えると、厚さが異なる微粒子２を用いることでもある。すなわち、範囲Ｃよりも範囲Ｄの方が厚さが大きい微粒子２の微粒子層が形成されていると言える。 On the other hand, the region 12 is enlarged, for example, in FIG. 1C, showing an example in which fine particles 2 having different particle diameters are used. On the substrate 1, fine particles 2 having a larger particle size in the range D than in the range C are solid-phased. For this reason, the area D of the metal layer 3 formed on the fine particles 2 is larger in the range D. Note that the use of fine particles 2 having different particle diameters means that fine particles 2 having different thicknesses are used. That is, it can be said that the fine particle layer of the fine particles 2 having a larger thickness in the range D than in the range C is formed.
他方、領域１３を拡大したのが、例えば図２（ｄ）であり、粒径が同じ微粒子２が用いられているが、微粒子２上に形成された金属層３及び金属層６３について、各々厚さが異なる領域が存在する。Ａ１及びＢ１の範囲に形成された金属層３は単一の金属元素Ａで構成され、Ｃ１及びＤ１の範囲に形成された金属層６３は金属元素Ａとは異なる単一の金属元素Ｂで構成されている。範囲Ａ１に存在する微粒子と範囲Ｂ１に存在する微粒子２の粒径は同じであるが、金属層３の厚さは範囲Ａ１よりも範囲Ｂ１の方が厚く形成される。また、範囲Ａ２に存在する微粒子２と範囲Ｂ２に存在する微粒子２の粒径は同じであるが、金属層６３の厚さは範囲Ａ２よりも範囲Ｂ２の方が厚く形成される。このように、異なる種類の単一金属元素からなる金属層の領域毎に、金属層の厚さが異なる領域がさらに形成される。 On the other hand, for example, FIG. 2D is an enlarged view of the region 13, and the fine particles 2 having the same particle diameter are used. The metal layer 3 and the metal layer 63 formed on the fine particles 2 are thicker. There are areas with different lengths. The metal layer 3 formed in the range of A1 and B1 is composed of a single metal element A, and the metal layer 63 formed in the range of C1 and D1 is composed of a single metal element B different from the metal element A Has been. The particle diameters of the fine particles existing in the range A1 and the fine particles 2 existing in the range B1 are the same, but the thickness of the metal layer 3 is larger in the range B1 than in the range A1. In addition, the particle diameters of the fine particles 2 existing in the range A2 and the fine particles 2 existing in the range B2 are the same, but the thickness of the metal layer 63 is larger in the range B2 than in the range A2. Thus, for each region of the metal layer composed of different types of single metal elements, a region having a different thickness of the metal layer is further formed.
図２（ｅ）は、図２（ｄ）とは異なる形態で、異なる種類の単一金属元素からなる金属層の領域毎に、金属層の厚さが異なる領域が形成されることを示す。図２（ｅ）は、領域１４を拡大した状態を示しており、粒径が異なる微粒子の領域が交互に形成されている。基板１上には、範囲Ｃ１よりも範囲Ｄ１の方が粒径が大きい微粒子２が固相化されている。また、範囲Ｃ２よりも範囲Ｄ２の方が粒径が大きい微粒子２が固相化されている。Ｃ１及びＤ１の範囲に形成された金属層３は単一の金属元素Ａで構成され、Ｃ２及びＤ２の範囲に形成された金属層６３は金属元素Ａとは異なる単一の金属元素Ｂで構成されている。微粒子２上に形成される金属層３の面積は範囲Ｃ１よりも範囲Ｄ１の方が大きくなる。また、微粒子２上に形成される金属層６３の面積は範囲Ｃ２よりも範囲Ｄ２の方が大きくなる。 FIG. 2E shows that a region having a different metal layer thickness is formed for each region of a metal layer made of a single metal element of a different type in a form different from that in FIG. FIG. 2E shows a state where the region 14 is enlarged, and fine particle regions having different particle diameters are alternately formed. On the substrate 1, fine particles 2 having a larger particle diameter in the range D1 than in the range C1 are solid-phased. Further, the fine particles 2 having a larger particle diameter in the range D2 than in the range C2 are solid-phased. The metal layer 3 formed in the range of C1 and D1 is composed of a single metal element A, and the metal layer 63 formed in the range of C2 and D2 is composed of a single metal element B different from the metal element A Has been. The area of the metal layer 3 formed on the fine particles 2 is larger in the range D1 than in the range C1. Further, the area of the metal layer 63 formed on the fine particles 2 is larger in the range D2 than in the range C2.
ここで、金属層３、６３で被覆されている微粒子２の表面に、例えば検体を吸着すれば、表面増強効果により、ラマン分光法、蛍光測定法、ＭＡＬＤＩ−ＴＯＦ（Matrix Assisted Laser Desorption Ionization−Time Of Flight）質量分析等様々な光学的分析方法の感度向上に寄与する。分析方法によって使用する波長領域が異なるため、金属層の組成、金属層のサイズ、金属層の形状等の最適条件が異なる。 Here, if, for example, a specimen is adsorbed on the surface of the fine particles 2 covered with the metal layers 3 and 63, Raman spectroscopy, fluorescence measurement, MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time) is obtained due to the surface enhancement effect. Of Flight) Contributes to improving the sensitivity of various optical analysis methods such as mass spectrometry. Since the wavelength region to be used differs depending on the analysis method, optimum conditions such as the composition of the metal layer, the size of the metal layer, and the shape of the metal layer are different.
本発明では、複数の分析手法に対して最適化された金属層が形成された金属付着微粒子１０を、図１のように、１枚の基板上に形成することができる。それぞれの手法に適した領域の表面積は減少するが、表面増強効果による感度向上率は、数十倍（蛍光及び赤外分光）から１００万倍以上（ラマン分光）であることから、それぞれの分析方法に対する信号の絶対強度は飛躍的に向上する。 In the present invention, the metal-attached fine particles 10 on which metal layers optimized for a plurality of analysis methods are formed can be formed on a single substrate as shown in FIG. Although the surface area of the region suitable for each method is reduced, the sensitivity improvement rate due to the surface enhancement effect is several tens of times (fluorescence and infrared spectroscopy) to over one million times (Raman spectroscopy). The absolute strength of the signal for the method is dramatically improved.
以上のように、本発明の分析用基板は、金属が付着した微粒子が基板上に形成されており、固相化微粒子が形成された領域は、光学的分析方法の種類に対応して、領域１１、１２のように、金属の種類もしくは金属の付着量を変えた領域、又は前記金属の種類及び金属の付着量を変えた領域に分割されている。 As described above, in the analysis substrate of the present invention, fine particles to which metal is attached are formed on the substrate, and the region where the solid-phased fine particles are formed corresponds to the type of optical analysis method. 11 and 12, it is divided into a region where the type of metal or the amount of deposited metal is changed, or a region where the type of metal and the amount of deposited metal are changed.
図３には、図１（ｂ）のように、金属層３の膜厚を領域毎に変化させる方法を示す。図３（ａ）のように、基板１上にシリカもしくはポリスチレン等からなる微粒子２を単層に分散して吸着させ、この微粒子２の上方の一部にマスク２０を配置する。ここで、微粒子２は基板１上に多層に吸着させるようにしても良い。基板１の表面に単層高密度に微粒子２を吸着させる具体的方法は任意であるが、例えば、以下のような方法で製造することができる。まず、微粒子溶液に、ＥＤＣ（１−Ｅｔｈｙｌ−３−［３−Ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ］ ｃａｒｂｏｄｉｉｍｉｄｅ Ｈｙｄｒｏｃｈｌｏｒｉｄｅ）やＮａＣｌなどの塩を添加（０．５Ｍ以下、好ましくは１００ｍＭ以下）し、混合溶液を作製する。例えば、微粒子溶液１溶に対してＥＤＣ溶液２溶を加える。この混合溶液を、基板１の表面へ塗布する。この際、基板１をガラスやプラスチックにて形成した場合には、この基板１にベース層を蒸着またはスパッタリングにより形成して、このベース層の表面へ混合溶液を塗布する。あるいは、基板１をシリコン等の金属にて形成した場合には、ベース層を形成することなく、この基板１の表面へ混合溶液を塗布する。そして、この基板１を数秒以上放置した後に蒸留水で洗浄して乾燥させ、この基板１の表面に単層高密度に微粒子２を吸着させる。 FIG. 3 shows a method of changing the film thickness of the metal layer 3 for each region as shown in FIG. As shown in FIG. 3A, fine particles 2 made of silica, polystyrene, or the like are dispersed and adsorbed on a substrate 1 in a single layer, and a mask 20 is disposed on a part above the fine particles 2. Here, the fine particles 2 may be adsorbed in multiple layers on the substrate 1. Although a specific method for adsorbing the fine particles 2 on the surface of the substrate 1 with a single-layer high density is arbitrary, for example, it can be produced by the following method. First, a salt solution such as EDC (1-Ethyl-3- [3-dimethylaminopropyl) carbohydrate) or NaCl is added to a fine particle solution (0.5 M or less, preferably 100 mM or less) to prepare a mixed solution. For example, the EDC solution 2 is added to the fine particle solution 1 solution. This mixed solution is applied to the surface of the substrate 1. At this time, when the substrate 1 is formed of glass or plastic, a base layer is formed on the substrate 1 by vapor deposition or sputtering, and the mixed solution is applied to the surface of the base layer. Alternatively, when the substrate 1 is formed of a metal such as silicon, the mixed solution is applied to the surface of the substrate 1 without forming a base layer. Then, the substrate 1 is allowed to stand for several seconds or more, then washed with distilled water and dried, and the fine particles 2 are adsorbed on the surface of the substrate 1 at a high density in a single layer.
この状態で蒸着法又はスパッタ法により、金、銀、プラチナ等の金属粒子２１を発生させることにより、図３（ｂ）のように、マスク２０で覆われた領域の微粒子２には、金属が付着せず、一方、マスク２０で覆われていない領域に存在する微粒子２の表面に金属層３ａが形成される。 In this state, metal particles 21 such as gold, silver, and platinum are generated by a vapor deposition method or a sputtering method, so that the fine particles 2 in the region covered with the mask 20 have a metal as shown in FIG. On the other hand, the metal layer 3 a is formed on the surface of the fine particles 2 present in the region that is not attached and is not covered with the mask 20.
次に、図３（ｃ）のように、マスク２０を取り除いた状態で２回目の蒸着又はスパッタを行うと、金属粒子２１が全体の微粒子２上に堆積し、図３（ｂ）で既に形成された金属層３ａに重ねて金属層が堆積される。したがって、図３（ｄ）のように、範囲Ａの金属層３の厚さは範囲Ｂの金属層３よりも薄くなる。なお、図３（ａ）〜（ｄ）までの工程を複数回繰り返すようにしても良い。 Next, as shown in FIG. 3C, when the second vapor deposition or sputtering is performed with the mask 20 removed, the metal particles 21 are deposited on the entire fine particles 2 and are already formed in FIG. 3B. A metal layer is deposited over the formed metal layer 3a. Therefore, as shown in FIG. 3D, the thickness of the metal layer 3 in the range A is thinner than the metal layer 3 in the range B. Note that the steps of FIGS. 3A to 3D may be repeated a plurality of times.
図３の製造工程において、例えば四角形の開口部を有するマスクを用いた場合、そのマスクパターンと領域毎の形成状態を示すのが、図１０である。図１０（ａ）は、四角形の開口部３４ａと遮蔽部３４ｂからなるマスク２０を上から見た平面図を示す。図３の製造工程に示すように、図１０（ａ）のマスクを用いて、微粒子上に第１回目の蒸着を行い、次にマスクを取り除いて第２回目の蒸着を行う。図１０（ｂ）は、第２回目の蒸着後に形成された２種類の領域を示す。図１０（ｂ）において、範囲Ａの金属層の厚さは範囲Ｂの金属層よりも薄くなる。 In the manufacturing process of FIG. 3, for example, when a mask having a square opening is used, FIG. 10 shows the mask pattern and the formation state of each region. FIG. 10A is a plan view of the mask 20 including the rectangular opening 34a and the shielding part 34b as viewed from above. As shown in the manufacturing process of FIG. 3, the first deposition is performed on the fine particles using the mask of FIG. 10A, and then the second deposition is performed after removing the mask. FIG. 10B shows two types of regions formed after the second deposition. In FIG. 10B, the thickness of the metal layer in the range A is thinner than the metal layer in the range B.
上記のように、金属層３の膜厚が領域毎に異なる金属付着微粒子１０を１枚の共通の基板上に形成することができる。マスク２０の除去前の金属粒子２１と、マスク２０の除去後の金属粒子２１とを異なる種類の金属粒子とし、範囲Ａと範囲Ｂの領域毎に異なる種類の金属を堆積することもできる。 As described above, the metal-attached fine particles 10 having different thicknesses of the metal layer 3 for each region can be formed on one common substrate. The metal particles 21 before the removal of the mask 20 and the metal particles 21 after the removal of the mask 20 may be different types of metal particles, and different types of metals may be deposited for each of the regions A and B.
金属層３の膜厚を領域毎に変化させる他の方法を図４に示す。まず、図４（ａ）のように、基板１上にシリカもしくはポリスチレン等からなる微粒子２を一層に分散して吸着させる。ここで、微粒子２は基板１上に多層に吸着させるようにしても良い。吸着方法は、図３で述べた上記方法と同様である。 FIG. 4 shows another method for changing the film thickness of the metal layer 3 for each region. First, as shown in FIG. 4A, fine particles 2 made of silica, polystyrene, or the like are dispersed and adsorbed on a substrate 1 in a single layer. Here, the fine particles 2 may be adsorbed in multiple layers on the substrate 1. The adsorption method is the same as the method described above with reference to FIG.
次に、図４（ｂ）のように、蒸着法又はスパッタ法により、金属粒子２１を発生させ、基板１上の微粒子２全体に同じ厚さに金属層３ｂを堆積する。図４（ｃ）のように微粒子２の上方の一部に、マスク２０を配置する。そして反応性イオンエッチング等のドライエッチング２２を行うことにより、マスク２０で覆われていない範囲の金属層３ｂは削られて薄くなり、一方、マスク２０で覆われた範囲の金属層３ｂは削られない。 Next, as shown in FIG. 4B, metal particles 21 are generated by vapor deposition or sputtering, and the metal layer 3b is deposited on the entire fine particles 2 on the substrate 1 to the same thickness. As shown in FIG. 4C, a mask 20 is disposed on a part above the fine particles 2. Then, by performing dry etching 22 such as reactive ion etching, the metal layer 3b in a range not covered with the mask 20 is shaved and thinned, while the metal layer 3b in a range covered with the mask 20 is shaved. Absent.
したがって、範囲Ａの金属層３の厚さは範囲Ｂの金属層３よりも薄くなる。なお、図４（ａ）〜（ｄ）までの工程を複数回繰り返すようにしても良い。このように、エッチング処理の際に、マスク２０を用いることにより、限定された領域に存在する金属層の膜厚を薄くすることができ、領域によって金属層の厚さが異なる金属付着微粒子１０を形成することができる。 Therefore, the metal layer 3 in the range A is thinner than the metal layer 3 in the range B. Note that the steps from FIGS. 4A to 4D may be repeated a plurality of times. In this way, the thickness of the metal layer existing in the limited region can be reduced by using the mask 20 during the etching process, and the metal-attached fine particles 10 having different metal layer thicknesses depending on the region can be obtained. Can be formed.
以上の方法をまとめると、まず、単分散のポリスチレン微粒子もしくはシリカ微粒子等を基板に単層もしくは多層として吸着させる。次に、第１回目の真空蒸着又はスパッタにより、金属層を微粒子上に形成することにより、金属付着微粒子を形成する。この方法により、基板上に金属付着微粒子を容易に形成することができる。次に、第２回目の真空蒸着工程又はスパッタ工程でマスクを用いて一部の微粒子のみに金属層を堆積させるか、又は金属層の一部を削除する手法を用いる。この手法により、異なる特性の金属付着微粒子を１枚の基板上に形成することができる。 To summarize the above method, first, monodisperse polystyrene fine particles or silica fine particles are adsorbed on a substrate as a single layer or a multilayer. Next, metal adhesion fine particles are formed by forming a metal layer on the fine particles by first vacuum deposition or sputtering. By this method, metal-attached fine particles can be easily formed on the substrate. Next, a technique is used in which a metal layer is deposited only on a part of the fine particles using a mask in the second vacuum vapor deposition process or sputtering process, or a part of the metal layer is deleted. By this method, metal-attached fine particles having different characteristics can be formed on one substrate.
次に、金属層３のサイズを領域毎に変化させる方法を説明する。作製方法は図４（ａ）、図４（ｂ）の工程とほぼ同じである。違いは、基板１上に分散して吸着させる微粒子２の粒径が領域毎に異なることである。領域毎に異なる粒径の微粒子２を分散吸着させた後、蒸着法又はスパッタ法により、一様に金属粒子２１を堆積させれば、粒径の大きな微粒子上には直径が大きい金属層が、粒径の小さな微粒子上には直径の小さい金属層が形成される。このようにして、図１（ｃ）の範囲Ｃ、範囲Ｄに示すように、金属層３のサイズを領域毎に変化させることができる。 Next, a method for changing the size of the metal layer 3 for each region will be described. The manufacturing method is almost the same as the process of FIGS. 4 (a) and 4 (b). The difference is that the particle diameters of the fine particles 2 dispersed and adsorbed on the substrate 1 differ from region to region. After the fine particles 2 having different particle diameters are dispersed and adsorbed for each region, and the metal particles 21 are uniformly deposited by vapor deposition or sputtering, a metal layer having a large diameter is formed on the fine particles having large particle diameters. A metal layer having a small diameter is formed on fine particles having a small particle diameter. In this way, the size of the metal layer 3 can be changed for each region as shown in the range C and the range D in FIG.
図５には、図３、４で用いるマスク２０のパターンが４種類例示されている。このマスクにより、様々な形状の領域が生じる。図５（ａ）には四角形の開口部３０ａと遮蔽部３０ｂとを有するマスク２０が示されている。図５（ｂ）には、三角形の開口部３１ａと遮蔽部３１ｂとを有するマスク２０が示されている。図５（ｃ）には、円形の開口部３２ａと遮蔽部３２ｂを有するマスク２０が示されている。開口部の形状については、図５（ａ）〜図５（ｃ）に示す形状以外の形状であっても良い。また、図５（ｄ）のように、遮蔽部３３ｂに異なる形状の開口部３３ａを配置するように構成したマスク２０であっても良い。開口部の形状だけではなく、開口部と遮蔽部との面積比率等もこれらに限られるものではなく、適宜変更しても良い。さらに、開口部の形状、サイズ、間隔は均一でなくても良い。 FIG. 5 illustrates four types of patterns of the mask 20 used in FIGS. This mask produces regions of various shapes. FIG. 5A shows a mask 20 having a square opening 30a and a shielding part 30b. FIG. 5B shows a mask 20 having a triangular opening 31a and a shielding part 31b. FIG. 5C shows the mask 20 having a circular opening 32a and a shielding part 32b. About the shape of an opening part, shapes other than the shape shown to Fig.5 (a)-FIG.5 (c) may be sufficient. Further, as shown in FIG. 5D, the mask 20 may be configured such that openings 33a having different shapes are arranged in the shielding part 33b. Not only the shape of the opening, but also the area ratio between the opening and the shielding part is not limited to these, and may be changed as appropriate. Furthermore, the shape, size, and spacing of the openings need not be uniform.
前述したように、図３の工程で、マスク２０の除去前の金属粒子２１と、マスク２０の除去後の金属粒子２１とを異なる種類の金属粒子として、領域毎に異なる種類の金属を堆積させることもできるが、領域毎に異なる種類の金属を微粒子２上に堆積させる他の方法を図６〜図７に示す。図６（ａ）に示すように、基板１上に微粒子２を一様に分散、吸着させる。この吸着方法は、図３での説明と同様である。マスク２０を微粒子２上に配置し、蒸着法又はスパッタ法により第１の金属粒子２１ａを堆積させる。図６（ｂ）のように、マスク２０の開口部に対応した微粒子２上には、第１の金属粒子２１ａによる金属層３が形成される。 As described above, in the process of FIG. 3, the metal particles 21 before the removal of the mask 20 and the metal particles 21 after the removal of the mask 20 are made to be different types of metal particles, and different types of metals are deposited in each region. However, other methods for depositing different types of metal on the fine particles 2 for each region are shown in FIGS. As shown in FIG. 6A, the fine particles 2 are uniformly dispersed and adsorbed on the substrate 1. This adsorption method is the same as described in FIG. The mask 20 is disposed on the fine particles 2 and the first metal particles 21a are deposited by vapor deposition or sputtering. As shown in FIG. 6B, the metal layer 3 made of the first metal particles 21 a is formed on the fine particles 2 corresponding to the openings of the mask 20.
次に、図７（ｃ）に示すように、マスク２０を矢印の方向（左方向）にずらした後、金属粒子２１ａとは異なる種類の金属粒子２１ｂを蒸着法又はスパッタ法により発生させ、微粒子２上に堆積させる。図７（ｄ）に示すように、マスク２０の開口部に相当する微粒子２には金属粒子２１ｂが堆積するが、遮蔽されている部分は、金属粒子２１ｂの堆積がなく、金属層３のままである。マスク２０の開口部に相当する微粒子２については、金属粒子２１ｂによる金属層６３のみが形成されたものと、金属粒子２１ａによる金属層３の上にさらに金属層６３が積層された多層金属層７３が形成されたものが存在する。このようにして、領域毎に異種の金属を表面に堆積させた金属付着微粒子１０を形成することができる。 Next, as shown in FIG. 7C, after the mask 20 is shifted in the direction of the arrow (left direction), a metal particle 21b of a type different from the metal particle 21a is generated by a vapor deposition method or a sputtering method. 2 is deposited. As shown in FIG. 7D, the metal particles 21b are deposited on the fine particles 2 corresponding to the openings of the mask 20. However, the shielded portion is not deposited with the metal particles 21b and remains the metal layer 3. It is. For the fine particles 2 corresponding to the openings of the mask 20, only the metal layer 63 made of the metal particles 21b is formed, and the multilayer metal layer 73 in which the metal layer 63 is further laminated on the metal layer 3 made of the metal particles 21a. Is formed. In this way, the metal-attached fine particles 10 in which different kinds of metals are deposited on the surface for each region can be formed.
図８は、例えば、六角形の開口部を有するマスクを用いた場合に、微粒子２上に異種金属が形成される過程を示す図である。マスクの上面から見た平面図を示す。実線で囲んだ六角形の開口部が、最初にマスクが配置された位置を示す。この状態で、第１の金属粒子を発生させて微粒子２上に金属層３を形成する。次に、マスクを図のマスク移動方向となる矢印方向に移動させて、破線で示す六角形の位置まで移動させる。 FIG. 8 is a diagram illustrating a process in which a dissimilar metal is formed on the fine particles 2 when, for example, a mask having a hexagonal opening is used. The top view seen from the upper surface of the mask is shown. A hexagonal opening surrounded by a solid line indicates a position where the mask is first arranged. In this state, first metal particles are generated to form the metal layer 3 on the fine particles 2. Next, the mask is moved in the direction of the arrow, which is the mask movement direction in the figure, and moved to the hexagonal position indicated by the broken line.
ここで、第２の金属粒子を発生させて、微粒子２上に金属層６３を形成する。すると、実線の六角形と破線の六角形とが重なる領域（斜線の領域）５２は、金属層３上に金属層６３が積層された多層金属層７３が形成される。一方、点々で示された領域５０は、金属層３が形成され、破線の六角形の白抜きの領域５１には、金属層６３が形成される。また、上下の六角形の間の領域５３には、全く金属層が形成されない。このように、異種の金属層が各々一層のみ形成された領域と、異種の金属層が積層された領域と、何も金属層が堆積されない領域が形成される。なお、マスクの移動方向は、図の方向ではなく、どの方向に移動させても良い。 Here, second metal particles are generated to form the metal layer 63 on the fine particles 2. Then, in a region (shaded region) 52 where the solid hexagon and the broken hexagon overlap each other, a multilayer metal layer 73 in which the metal layer 63 is laminated on the metal layer 3 is formed. On the other hand, the metal layer 3 is formed in the region 50 indicated by dots, and the metal layer 63 is formed in the hexagonal white region 51 of the broken line. Also, no metal layer is formed in the region 53 between the upper and lower hexagons. In this way, a region where only one different metal layer is formed, a region where different metal layers are stacked, and a region where no metal layer is deposited are formed. Note that the moving direction of the mask may be moved in any direction, not the direction shown in the figure.
図９には、六角形の開口部を有するマスクを利用し、領域毎に異種金属を堆積した金属付着微粒子が形成されたサンプルの走査型顕微鏡写真像を示す。これは、六角形の開口部を有するマスクを用いて金を蒸着させ、次に、マスクを所定の方向に移動させた後、銀を蒸着したものである。基板上には粒径１００ｎｍのシリカ微粒子が均一に吸着されており、領域４０には厚さ２０ｎｍの金、領域４１には厚さ２０ｎｍの銀、領域４２には厚さがそれぞれ２０ｎｍの金と厚さ２０ｎｍの銀とが積層された薄膜が形成されている。領域４３は全く金属が形成されていない領域であり、このような領域が存在しても良い。 FIG. 9 shows a scanning micrograph image of a sample in which metal-adhered fine particles in which different kinds of metal are deposited in each region are formed using a mask having a hexagonal opening. In this method, gold is vapor-deposited using a mask having a hexagonal opening, and then silver is vapor-deposited after the mask is moved in a predetermined direction. Silica fine particles having a particle diameter of 100 nm are uniformly adsorbed on the substrate, the region 40 is 20 nm thick gold, the region 41 is 20 nm thick silver, and the region 42 is 20 nm thick gold. A thin film in which 20 nm thick silver is laminated is formed. The region 43 is a region where no metal is formed, and such a region may exist.
図６〜図８までに示したように、１種類のマスクを所定の方向に移動させることにより、２種類の光学特性の異なる領域を形成する方法の他の例を図１１に示す。図１１（ａ）は、四角形の開口部３５ａと遮蔽部３５ｂからなるマスク２０を上から見た平面図を示す。マスクの横方向における開口部３５ａの間隔は、開口部３５ａの１つ分開けて配置されている。図１１（ｂ）は、２種類の光学特性の異なる領域の形成状態を示す。まず、図１１（ａ）のマスクを用いて、微粒子上に第１回目の金属元素の蒸着を行い、次にマスクを図１１（ａ）の矢印に示すマスクの移動方向（横方向）に、開口部３５ａの間隔分移動させる。その状態で、微粒子上に第２回目の金属元素の蒸着を行う。例えば、第１回目の蒸着と第２回目の蒸着とで金属元素の種類を変えれば、図１１（ｂ）に示すように、２種類の光学特性の異なる領域Ｘ、Ｙを形成することができる。 As shown in FIGS. 6 to 8, FIG. 11 shows another example of a method of forming two types of regions having different optical characteristics by moving one type of mask in a predetermined direction. Fig.11 (a) shows the top view which looked at the mask 20 which consists of a square opening part 35a and the shielding part 35b from the top. The interval between the openings 35a in the lateral direction of the mask is arranged to be one opening 35a apart. FIG. 11B shows the formation state of two regions having different optical characteristics. First, using the mask of FIG. 11A, the first metal element is vapor-deposited on the fine particles, and then the mask is moved in the mask moving direction (lateral direction) indicated by the arrow in FIG. It is moved by the interval of the opening 35a. In this state, a second metal element is deposited on the fine particles. For example, if the type of metal element is changed between the first vapor deposition and the second vapor deposition, two types of regions X and Y having different optical characteristics can be formed as shown in FIG. .
次に、図１２は、図６〜図８と同様、１種類のマスクで光学特性の異なる３種類の領域を形成する例を示す。図１２（ａ）は、四角形の開口部３６ａと遮蔽部３６ｂからなるマスク２０を上から見た平面図を示す。図１２（ｂ）は、３種類の光学特性の異なる領域の形成状態を示す。まず、図１２（ａ）のマスクを用いて、微粒子上に第１回目の金属元素Ａの蒸着を行い、次にマスクを図１２（ａ）の矢印に示すマスクの移動方向（横方向）に、少し移動させる。その状態で、微粒子上に第２回目の金属元素Ｂの蒸着を行う。図１２（ｂ）に示すように、金属元素Ａが堆積された領域Ｘ、金属元素Ｂが堆積された領域Ｙ、金属元素Ａと金属元素Ｂとが堆積された領域Ｗの３種類の光学特性の異なる領域が形成される。 Next, FIG. 12 shows an example in which three types of regions having different optical characteristics are formed with one type of mask, as in FIGS. FIG. 12A shows a plan view of the mask 20 including the rectangular opening 36a and the shielding part 36b as viewed from above. FIG. 12B shows the formation state of three regions having different optical characteristics. First, using the mask of FIG. 12A, the first deposition of the metal element A is performed on the fine particles, and then the mask is moved in the mask movement direction (lateral direction) indicated by the arrow in FIG. Move a little. In this state, a second metal element B is deposited on the fine particles. As shown in FIG. 12B, there are three types of optical characteristics: a region X where the metal element A is deposited, a region Y where the metal element B is deposited, and a region W where the metal element A and the metal element B are deposited. Different regions are formed.
次に、図１３は、１種類のマスクで光学特性の異なる３種類の領域を形成する他の例を示す。図１３（ａ）は、円形の開口部３７ａと遮蔽部３７ｂからなるマスク２０を上から見た平面図を示す。図１３（ｂ）は、３種類の光学特性の異なる領域の形成状態を示す。まず、図１３（ａ）のマスクを用いて、微粒子上に第１回目の金属元素Ａの蒸着を行い、次にマスクを図１３（ａ）の矢印に示すマスクの第１の移動方向に、少し移動させる。その状態で、微粒子上に第２回目の金属元素Ｂの蒸着を行う。次に、マスクを図１３（ａ）の矢印に示すマスクの第２の移動方向に、少し移動させる。その状態で、微粒子上に第３回目の金属元素Ｃの蒸着を行う。図１３（ｂ）に示すように、金属元素Ａが堆積された領域Ｘ、金属元素Ｂが堆積された領域Ｙ、金属元素Ｃが堆積された領域Ｚの３種類の光学特性の異なる領域が形成される。この図１３の方法の利点は、マスクを一枚用いれば良いだけなので、工程時間の短縮が可能である。一方、欠点は基板の面積が無駄になり、面積の自由度がないことである。 Next, FIG. 13 shows another example in which three types of regions having different optical characteristics are formed with one type of mask. Fig.13 (a) shows the top view which looked at the mask 20 which consists of circular opening part 37a and shielding part 37b from the top. FIG. 13B shows the formation state of three regions having different optical characteristics. First, using the mask of FIG. 13A, a first metal element A is deposited on the fine particles, and then the mask is moved in the first moving direction of the mask indicated by the arrow in FIG. Move a little. In this state, a second metal element B is deposited on the fine particles. Next, the mask is slightly moved in the second movement direction of the mask indicated by the arrow in FIG. In this state, the third metal element C is deposited on the fine particles. As shown in FIG. 13B, three types of regions having different optical characteristics are formed: a region X where the metal element A is deposited, a region Y where the metal element B is deposited, and a region Z where the metal element C is deposited. Is done. The advantage of the method of FIG. 13 is that only one mask needs to be used, so that the process time can be shortened. On the other hand, the disadvantage is that the area of the substrate is wasted and there is no freedom of area.
次に、図１４は、複数のマスクを用いて、光学特性の異なる３種類の領域を形成する例を示す。図１４（ａ）は、円形の開口部３８０ａと遮蔽部３８０ｂからなる第１のマスクを上から見た平面図を示す。図１４（ｂ）は、円形の開口部３８１ａと遮蔽部３８１ｂからなる第２のマスクを上から見た平面図を示す。第１のマスクと第２のマスクは、開口部の大きさは同じであるが、開口部の配置位置が異なっている。図１４（ｃ）は、円形の開口部３８２ａと遮蔽部３８２ｂからなる第３のマスクを上から見た平面図を示す。第３のマスクは、第１のマスク及び第２のマスクとは、開口部の大きさも配置位置も異なっている。 Next, FIG. 14 shows an example in which three types of regions having different optical characteristics are formed using a plurality of masks. FIG. 14A shows a plan view of the first mask composed of a circular opening 380a and a shielding part 380b as seen from above. FIG. 14B is a plan view of the second mask composed of the circular opening 381a and the shielding part 381b as viewed from above. The first mask and the second mask have the same opening size, but are different in the arrangement position of the opening. FIG. 14C shows a plan view of the third mask composed of the circular opening 382a and the shielding part 382b as seen from above. The size of the opening and the arrangement position of the third mask are different from those of the first mask and the second mask.
図１４（ｂ）は、３種類の光学特性の異なる領域の形成状態を示す。まず、図１４（ａ）のマスクを用いて、微粒子上に第１回目の金属元素Ａの蒸着を行い、次に、図１４（ａ）のマスクを図１４（ｂ）のマスクと取り替えて、微粒子上に第２回目の金属元素Ｂの蒸着を行う。その後、図１４（ｂ）のマスクを図１４（ｃ）のマスクと取り替えて、微粒子上に第３回目の金属元素Ｃの蒸着を行う。図１４（ｄ）に示すように、金属元素Ａが堆積された領域Ｘ、金属元素Ｂが堆積された領域Ｙ、金属元素Ｃが堆積された領域Ｚの３種類の光学特性の異なる領域が形成される。この図１４の方法の利点は、基板の面積の無駄が省け、領域の面積及び形状の自由度が増すことである。一方、欠点は、複数のマスクが必要となり、作業工程時間がかかることである。 FIG. 14B shows the formation state of three regions having different optical characteristics. First, using the mask of FIG. 14A, the first metal element A is deposited on the fine particles, and then the mask of FIG. 14A is replaced with the mask of FIG. A second metal element B is deposited on the fine particles. Thereafter, the mask shown in FIG. 14B is replaced with the mask shown in FIG. 14C, and the third metal element C is deposited on the fine particles. As shown in FIG. 14D, three types of regions having different optical characteristics are formed: a region X where the metal element A is deposited, a region Y where the metal element B is deposited, and a region Z where the metal element C is deposited. Is done. The advantage of the method of FIG. 14 is that the area of the substrate is not wasted and the area and shape of the area are increased. On the other hand, a drawback is that a plurality of masks are required, and work process time is required.
次に、図１５は、複数のマスクを用いて、光学特性の異なる４種類の領域を形成する例を示す。図１５（ａ）は、長方形の開口部３９０ａと遮蔽部３９０ｂからなる第１のマスクを上から見た平面図を示す。図１５（ｂ）は、長方形の開口部３９１ａと遮蔽部３９１ｂからなる第２のマスクを上から見た平面図を示す。第１のマスクと第２のマスクは、開口部の形状や大きさは同じであるが、開口部の配置位置が異なっている。また、第１のマスクと第２のマスクは、開口部位置が一部重なるように形成されている。図１５（ｃ）は、円形の開口部３９２ａと遮蔽部３９２ｂからなる第３のマスクを上から見た平面図を示す。 Next, FIG. 15 shows an example in which four types of regions having different optical characteristics are formed using a plurality of masks. FIG. 15A shows a plan view of a first mask composed of a rectangular opening 390a and a shielding part 390b as viewed from above. FIG. 15B is a plan view of the second mask composed of the rectangular opening 391a and the shielding part 391b as viewed from above. The first mask and the second mask have the same shape and size of the openings, but the positions of the openings are different. Further, the first mask and the second mask are formed so that the positions of the openings partially overlap. FIG. 15C is a plan view of the third mask including the circular opening 392a and the shielding part 392b as viewed from above.
図１５（ｄ）は、４種類の光学特性の異なる領域の形成状態を示す。まず、図１５（ａ）のマスクを用いて、微粒子上に第１回目の金属元素Ａの蒸着を行い、次に、図１５（ａ）のマスクを図１５（ｂ）のマスクと取り替えて、微粒子上に第２回目の金属元素Ｂの蒸着を行う。その後、図１５（ｂ）のマスクを図１５（ｃ）のマスクと取り替えて、微粒子上に第３回目の金属元素Ｃの蒸着を行う。図１５（ｄ）に示すように、金属元素Ａが堆積された領域Ｘ、金属元素Ｂが堆積された領域Ｙ、金属元素Ｃが堆積された領域Ｚ、金属元素Ａと金属元素Ｂとが堆積された領域Ｗの４種類の光学特性の異なる領域が形成される。 FIG. 15D shows the formation state of four regions having different optical characteristics. First, using the mask of FIG. 15A, the first metal element A is deposited on the fine particles, and then the mask of FIG. 15A is replaced with the mask of FIG. 15B. A second metal element B is deposited on the fine particles. Thereafter, the mask shown in FIG. 15B is replaced with the mask shown in FIG. 15C, and a third metal element C is deposited on the fine particles. As shown in FIG. 15D, the region X where the metal element A is deposited, the region Y where the metal element B is deposited, the region Z where the metal element C is deposited, the metal element A and the metal element B are deposited. Four types of regions having different optical characteristics are formed in the region W.
図１６は、光を用いた各分析法において最適条件が異なることを示すもので、ここでは、表面増強蛍光法および表面増強ラマン分光法を比較した。 FIG. 16 shows that the optimum conditions differ in each analysis method using light. Here, the surface-enhanced fluorescence method and the surface-enhanced Raman spectroscopy were compared.
図１６（ａ）は、表面増強蛍光法で測定した結果を示す。横軸は、銀薄膜の蒸着厚（ｎｍ）を、縦軸は増強率を示す。測定対象は以下のように構成した。基板上に粒径１０８ｎｍのシリカ微粒子を分散、吸着させ、シリカ微粒子上に銀（Ａｇ）薄膜を蒸着した。銀薄膜に濃度２μｇ／ｍｌの蛍光タンパク質（ＲＰＥ）を固定した。励起光波長５４３ｎｍ、検出波長５７０ｎｍで測定した。 FIG. 16 (a) shows the results measured by the surface enhanced fluorescence method. The horizontal axis represents the deposition thickness (nm) of the silver thin film, and the vertical axis represents the enhancement rate. The measurement object was configured as follows. Silica fine particles having a particle diameter of 108 nm were dispersed and adsorbed on the substrate, and a silver (Ag) thin film was deposited on the silica fine particles. Fluorescent protein (RPE) at a concentration of 2 μg / ml was immobilized on the silver thin film. Measurement was performed at an excitation light wavelength of 543 nm and a detection wavelength of 570 nm.
このとき、銀蒸着厚を基板毎に、５、１０、１５、２０、２５ｎｍと変えて測定した結果が図１６（ａ）に示されている。この測定結果より、銀蒸着厚１５ｎｍのときに、最も増強率が高くなっており、表面増強蛍光法の場合における銀膜厚の最適条件は、１５ｎｍであることがわかる。 FIG. 16A shows the result of measurement when the silver deposition thickness was changed to 5, 10, 15, 20, and 25 nm for each substrate at this time. From this measurement result, it can be seen that the enhancement rate is the highest when the silver deposition thickness is 15 nm, and the optimum condition of the silver film thickness in the case of the surface enhanced fluorescence method is 15 nm.
図１６（ｂ）は、表面増強ラマン分光法による測定結果を示す。横軸は銀薄膜の蒸着厚（ｎｍ）を、縦軸はラマン信号強度を示す。測定対象は以下のように構成した。基板上に粒径１００ｎｍのシリカ微粒子を分散、吸着させ、シリカ微粒子上にシリカ微粒子上に銀（Ａｇ）薄膜を蒸着した。銀薄膜に染料の一種であるＢＰＥを６５ｎＭ固定し、励起光波長５３２ｎｍ、検出ピーク１６１６ｃｍ−１で測定した。 FIG. 16 (b) shows the measurement results by surface enhanced Raman spectroscopy. The horizontal axis represents the deposition thickness (nm) of the silver thin film, and the vertical axis represents the Raman signal intensity. The measurement object was configured as follows. Silica fine particles having a particle diameter of 100 nm were dispersed and adsorbed on the substrate, and a silver (Ag) thin film was deposited on the silica fine particles on the silica fine particles. The silver thin film was fixed with 65 nM of BPE, which is a kind of dye, and measured at an excitation light wavelength of 532 nm and a detection peak of 1616 cm −1 .
このとき、銀蒸着厚を基板毎に、１０〜７０ｎｍまで１０ｎｍ間隔で変えて７点測定した結果が図１６（ｂ）に示されている。この測定結果からわかるように、銀蒸着厚は、６０ｎｍのときに最もラマン信号強度が大きくなっており、表面増強ラマン分光法の場合における銀膜厚の最適条件は、６０ｎｍであることがわかる。 At this time, the result of measuring seven points by changing the silver deposition thickness for each substrate from 10 to 70 nm at intervals of 10 nm is shown in FIG. As can be seen from the measurement results, the Raman signal intensity is greatest when the silver deposition thickness is 60 nm, and the optimum condition of the silver film thickness in the case of surface enhanced Raman spectroscopy is 60 nm.
そこで、表面増強蛍光法および表面増強ラマン分光法の両方の分析方法に適した基板として、厚さ１５ｎｍと６０ｎｍの銀薄膜が形成された基板を作製することが望ましいことがわかる。ちなみに、増強率は励起光波長及びサンプルの吸収スペクトルに依存することから、測定対象と励起光波長により付着させる金属の種類又は／及び金属の付着量を決定し、これらの異なる領域を有する基板を作製することになる。これらの領域を作製する方法は、既に説明した前述の方法を用いれば良い。 Therefore, it can be seen that it is desirable to produce a substrate on which a silver thin film having a thickness of 15 nm and 60 nm is formed as a substrate suitable for both the surface-enhanced fluorescence method and the surface-enhanced Raman spectroscopic analysis method. Incidentally, since the enhancement factor depends on the excitation light wavelength and the absorption spectrum of the sample, the type of metal to be attached and / or the amount of metal attached is determined according to the measurement object and the excitation light wavelength, and the substrate having these different regions is determined. Will be produced. As the method for manufacturing these regions, the above-described method may be used.
本発明の分析用基板及び分析用基板の製造方法は、表面増強蛍光法、表面増強ラマン分光法、表面プラズモン共鳴による分析、局在型表面プラズモン共鳴による分析、質量分析等、表面増強効果を利用した光学的分析方法に幅広く適用することができる。 The analytical substrate and analytical substrate manufacturing method of the present invention utilize surface enhancement effects such as surface-enhanced fluorescence, surface-enhanced Raman spectroscopy, analysis by surface plasmon resonance, analysis by localized surface plasmon resonance, and mass spectrometry. It can be widely applied to the optical analysis method.
１ 基板
２ 微粒子
３ａ 金属層
３ｂ 金属層
３ 金属層
１０ 金属付着微粒子
２０ マスク
２１ 金属粒子
２１ａ 金属粒子
２１ｂ 金属粒子
DESCRIPTION OF SYMBOLS 1 Substrate 2 Fine particle 3a Metal layer 3b Metal layer 3 Metal layer 10 Metal adhesion fine particle 20 Mask 21 Metal particle 21a Metal particle 21b Metal particle
Claims (6)
表面増強効果を利用した光学的分析方法の種類に対応して、前記微粒子の上に付着させる金属量を前記微粒子の固相化領域毎に変える第２の工程とを備えたことを特徴とする分析用基板の製造方法。 A first step of solidifying fine particles on a substrate;
In accordance with the type of optical analysis method using the surface enhancement effect, a second step of changing the amount of metal deposited on the fine particles for each solid-phase region of the fine particles is provided. A method for manufacturing an analytical substrate.
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